Integrated Chip Carriers With Thermocycler Interfaces And Methods Of Using The Same

ABSTRACT

Methods and systems are provided for conducting a reaction at a selected temperature or range of temperatures over time. An array device is provided. The array device contains separate reaction chambers and is formed as an elastomeric block from multiple layers. At least one layer has at least one recess that recess has at least one deflectable membrane integral to the layer with the recess. The array device has a thermal transfer device proximal to at least one of the reaction chambers. The thermal transfer device is formed to contact a thermal control source. Reagents for carrying out a desired reaction are introduced into the array device. The array device is contacted with a thermal control device such that the thermal control device is in thermal communication with the thermal control source so that a temperature of the reaction in at least one of the reaction chamber is changed as a result of a change in temperature of the thermal control source.

PRIORITY CLAIM

This application is a divisional of U.S. patent application Ser. No.11/740,735, filed Apr. 26, 2007, now allowed; which is a continuation ofU.S. patent application Ser. No. 11/058,106, filed Feb. 14, 2005; whichis a continuation-in-part of U.S. patent application Ser. No.11/043,895, filed Jan. 25, 2005; which claims priority under 35 U.S.C.§119(e) of U.S. Provisional Patent Application No. 60/558,316, filedMar. 30, 2004, U.S. Provisional Patent Application No. 60/557,715, filedMar. 29, 2004, and U.S. Provisional Patent Application No. 60/539,283,filed Jan. 25, 2004, each of which is herein incorporated by referencein its entirety for all purposes and the specific purposes disclosedherein.

CROSS-REFERENCES TO PATENTS AND PATENT APPLICATIONS

The invention is related to the subject matter disclosed in U.S. patentapplication Ser. No. 09/796,666, filed Feb. 28, 2001, issued as U.S.Pat. No. 6,408,878 on Jun. 25, 2002 (“Unger”); U.S. patent applicationSer. No. 09/887,997, filed Jun. 22, 2001, issued as U.S. Pat. No.7,052,545 on May 30, 2006 (“Hansen”); and U.S. patent application Ser.No. 10/160,906, filed May 30, 2002, now abandoned (“Delucas”), which isa continuation of U.S. patent application Ser. No. 09/543,326, filed onApr. 5, 2000, now abandoned, which claims priority to U.S. ProvisionalPatent Application No. 60,128,012, filed on Apr. 6, 1999, the disclosureof each of which is herein incorporated by reference for all purposes.

The invention is further related to U.S. patent application Ser. No.10/997,714, filed Nov. 24, 2004, now abandoned, which claims priority toU.S. Provisional Patent Application No. 60/525,245, filed Nov. 26, 2003,the disclosure of each of which is herein incorporated by reference forall purposes.

The invention is further related to U.S. patent application Ser. No.10/827,917, filed Apr. 19, 2004, issued as U.S. Pat. No. 7,279,146 onOct. 9, 2007, which claims priority to U.S. Provisional PatentApplication No. 60/509,098, filed Oct. 5, 2003, 60/466,305, filed Apr.28, 2003, and 60/463,778, filed Apr. 17, 2003, the disclosure of each ofwhich is herein incorporated by reference for all purposes.

FIELD OF THE INVENTION

This invention relates to the fields of microfluidics, lab-on-a-chip,and Polymerase Chain Reactions (“PCR”), biochemical analysis, proteincrystallization and screening for protein crystallization conditions,microfabrication, laboratory robotics, and automated biologicalscreening and analysis, among other fields.

BACKGROUND OF THE INVENTION

Crystallization is an important technique to the biological and chemicalarts. Specifically, a high-quality crystal of a target compound can beanalyzed by x-ray diffraction techniques to produce an accuratethree-dimensional structure of the target. This three-dimensionalstructure information can then be utilized to predict functionality andbehavior of the target.

In theory, the crystallization process is simple. A target compound inpure form is dissolved in solvent. The chemical environment of thedissolved target material is then altered such that the target is lesssoluble and reverts to the solid phase in crystalline form. This changein chemical environment is typically accomplished by introducing acrystallizing agent that makes the target material less soluble,although changes in temperature and pressure can also influencesolubility of the target material.

In practice however, forming a high quality crystal is generallydifficult and sometimes impossible, requiring much trial and error andpatience on the part of the researcher. Specifically, the highly complexstructure of even simple biological compounds means that they are notamenable to forming a highly ordered crystalline structure. Therefore, aresearcher must be patient and methodical, experimenting with a largenumber of conditions for crystallization, altering parameters such assample concentration, solvent type, countersolvent type, temperature,and duration in order to obtain a high quality crystal, if in fact acrystal can be obtained at all.

Accordingly, there is a need in the art for methods and structures forperforming high throughput screening of crystallization of targetmaterials.

Microfluidic devices are defined as devices having one or more fluidicpathways, often called channels, microchannels, trenches, or recesses,having a cross-sectional dimension below 1000 μm, and which offerbenefits such as increased throughput and reduction of reaction volumes.Interfacing microfluidic devices to macrosale systems, such as roboticliquid dispensing systems, has been challenging, often resulting in aloss of the number of reactions that can be carried out in parallel in asingle microfluidic device. As a non-limiting example, Delucasdiscloses, among other things, using a microfluidic device to conductnanoliter scale protein crystallization screening reactions in aparallel array format.

Unger discloses, among other things, microfluidic devices having anelastomeric block with a deflectable membrane. In one embodimentdisclosed, which is depicted in FIGS. 1A and 1B, first elastomeric layer1, having bottom surface 8 with microfabricated recess 2 formed therein,is bonded to top surface 7 of second elastomeric layer 3 havingmicrofabricated recess 4 formed therein, to form an elastomeric block 5having a first channel 6 formed from the recess 2 of the firstelastomeric layer 1 being closed off by top surface 7 of secondelastomeric layer 3, and where recess 4 of the second elastomeric layeris overlapped by first channel 6 formed, deflectable membrane 8 isformed by a portion of second elastomeric layer 3 separating firstchannel 6 from recess 4 of second elastomeric layer 3. Elastomeric block5 may then be attached to substrate 9 so that recess 4 of secondelastomeric layer 3 forms second channel 10 with a top surface ofsubstrate 9. Fluid flow through second channel 10 may be controlled byactuating deflectable membrane 8 to deflect into and out of secondchannel 10. Deflectable membrane 8 may be actuated by increasing ordecreasing the fluid pressure in first channel 6 to cause deflectablemembrane 8 to deflect into or out of second channel 10, respectively.Alternatively, by increasing or decreasing the fluid pressure in secondchannel 10, deflectable membrane 8 can be deflected into or out of firstchannel 6, respectively.

FIG. 1C depicts the use of the device just described wherein liquid isintroduced into second channel 10 through via 11, which was made bycoring a fluid path from the top of the elastomeric block through firstelastomeric layer 1 part of second elastomeric layer 3 into secondchannel 10. The fluid filling second channel 10 could then bepartitioned by applying fluid pressure, such as gas pressure, throughsecond via 13, which was made by coring through first elastomeric layer1 into first channel 6 so that when the pressure was increased in firstchannel 6, deflectable membrane 8 deflected down into second channel 10to contact the surface of substrate 9. Particular devices of Ungerprovide for high-density, reliable microfluidic devices in which themovement of fluid therein could be evoked and/or regulated by actuatingthe deflectable membrane to cause the membrane to function as part of avalve or pump.

An ideal application for microfluidic devices is screening forconditions that will cause a protein to form a crystal large enough forstructural analysis. Protein crystallization is an important step indetermining the structure of such proteins. Typically, reactions wereset up by manually pipetting a solution containing a protein and asolution containing a protein crystallization reagent to cause theprotein to form a crystal large enough to place in line with an X-raysource to perform X-ray diffraction studies. Determining the rightconditions that will form a large enough crystal is often determined byseemingly countless trial and error experiments. Consequently, preciousprotein isolates are exceedingly limited in supply and therefore need tobe judiciously used while screening for the right crystallizationconditions. As a way to spare protein consumption during conditionscreening, efforts were made to reduce the volume of proteincrystallization assays while increasing the number of experimentsperformed in parallel during the screen. Delucas discloses, among otherthings, methods and devices for carrying out nanoliter scale (nanoscale)protein crystallization experiments. In one embodiment disclosed, amicrofluidic device is used to carryout nanoscale proteincrystallization experiments in wells formed in a substrate.

Hansen discloses, among other things, microfluidic devices for carryingout protein crystallization reactions. Some of the embodiments disclosedin Hansen employ Unger's elastomeric block having deflectable membranestherein to regulate fluid flow. For example, a microfluidic devicehaving a first chamber containing a solution of a protein is in fluidcommunication with a second chamber containing a solution containing acrystallizing agent that when contacted with the protein in the firstchamber, may induce that protein to form crystals. In one example ofmany, the fluid communication between each chamber is through one ormore channels. A valve situated between each of the chambers and incommunication with the channel can be actuated to regulate the diffusionbetween the two chambers. The first chamber is in communication with afirst inlet for introducing the solution containing the protein into thefirst chamber, and the second chamber agent is in communication with asecond inlet for introducing the crystallization agent into thatchamber.

Hansen discloses, among other things, a carrier for holding themicrofluidic device of Hansen. An example of the Hansen carrier is shownin FIG. 2 where microfluidic structure 11000, which has several inletsand inlet rows such as well row 11012 a and well row 11012 b, sampleinlet 11012 c and containment valve control inlet 11012 d and interfacevalve control inlet 11012 e, is placed inside a frame base 11002 inreceiving area 1106 having view window 1103 therein. Top frame 11014,which has pressure cavities 11026 and 11024 is placed upon frame base11002 with microfluidic structure 11000 sandwiched between so that eachpressure cavities seals against well rows 11012 a and 11012 b to formpressure chambers on top of each well row. In use, each well in wellrows 11012 a and 11012 b are typically filled with different reagentsfor crystallizing proteins and sample inlet 11012 c is loaded with asample solution containing a protein to be crystallized. Containmentvalve control inlet 11012 d and interface valve control inlet 11012 eare typically filled with a liquid, such as an oil or water, tohydraulically actuate the valves in the microfluidic device. Pneumaticlines are inserted into control inlets 11012 d and 11012 e to applypressurized gas in fluidic communication with the liquid containedwithin each control inlet channel within the microfluidic device, whichin turn deflect membrane valve at certain intersections between thechannels of the first elastomeric layer and the second elastomericlayer, as shown in FIG. 1.

Likewise, sample solution can be driven into a channel and on intochambers inside the microfluidic device by similarly applying gaspressure to the sample inlet 11012 c to cause the sample solution todevelop hydraulic pressure to move it through the channel into thechambers. Reagents loaded into wells of well row 11012 a and 11012 b canalso be driven into their corresponding channels and on into chambersinside the microfluidic device by applying gas pressure to each of thepressure cavities. Once each of sample and reagent chambers within themicrofluidic device have been filled, containment valves may be thenclosed by actuating deflectable membranes in communication with theinlet channel preceding the chamber to keep the sample and reagentsinside their corresponding chambers. Meanwhile, an interface valvesbetween each of the sample/reagent chamber pairs is kept closed to keepthe reagent from diffusing into the sample and the sample from diffusinginto the reagent chambers. After the filling of all chambers iscomplete, free interface diffusion can begin by opening the interfacevalves, while keeping the containment valves closed.

Protein crystallization experiments performed using the devicesdisclosed in Hansen may take several days to perform. As mentioned, thecontainment valves must be kept closed at all time to prevent sample orreagents from moving out of the chambers, potentiallycross-contaminating each other. Accordingly, a source of pneumaticpressure to create a constant source of hydraulic pressure need bemaintained to keep the containment valves closed. This can be done byhaving an “umbilical cord” connecting the carrier connected to a sourceof gas pressure such as a regulated gas supply. However, such umbilicalcords may limit a user's ability to move a carrier about a laboratory,for example, into a refrigerator or incubator to achieve temperaturecontrol. Thus, there is a need for a system that would liberate amicrofluidic device, such as those described by Hansen or Unger, fromthe apparent need for an umbilical cord to maintain valve actuation.

Schulte et al. (“Schulte”), U.S. Patent Publication No. 2003-0034306 A1,published on Feb. 20, 2003, entitled “Well-Plate Microfluidics,” whichis hereby incorporated by reference for all purposes, disclosesmicrofluidic devices, however, there are numerous and substantialdifferences between the invention disclosed herein and the devices ofSchulte.

SUMMARY OF THE INVENTION

The present invention provides microfluidic devices and methods fortheir use. The invention further provides apparatus and systems forusing the microfluidic devices of the invention, analyze reactionscarried out in the microfluidic devices, and systems to generate, store,organize, and analyze data generated from using the microfluidicdevices. The invention further provides methods of using and makingmicrofluidic systems and devices which, in some embodiments, are usefulfor crystal formation.

The invention provides apparatus for operating a microfluidic device. Inone embodiment, the apparatus includes a platen having a platen facewith one or more fluid ports therein. The fluid ports spatiallycorrespond to one or more wells on a surface of the microfluidic device.A platform for holding the microfluidic device relative to the platen isincluded, and a platen actuator for urging the platen against themicrofluidic device so that at least one of the fluid ports of theplaten is urged against one of the wells to form a pressure chambercomprising the well and the port, so that when pressurized fluid isintroduced or removed into or from the pressure chamber through one ofthe ports, fluid pressure is changed therein.

In other embodiments, the apparatus includes a robotic platen actuator;the platen actuator is under electronic control by a controller; thecontroller is a computer or under computer control; the computer isfollowing a program; the program was customized by a user of theapparatus; the microfluidic device includes first and second chambers influid communication with each other through a channel and a valvedisposed along the channel which when opened or closed controls fluidcommunication between the first and second chambers, and wherein thevalve is under the control of an automated valve actuating device whenthe microfluidic device is coupled to the platen; the automated valveactuating device is further under computer control; the valve is openedand closed using the automated valve actuating device; the valvecomprises a deflectable membrane; and the platen actuator is adapted fordelivering a pressurized fluid to the at least one fluid pressure portusing a pressure between about one pound per square inch (1 psi) andabout thirty-five pounds per square inch (35 psi).

The present invention further provides for microfluidic systems. Onesuch system includes a microfluidic device having a plurality ofchambers, with the microfluidic device coupled to a carrier and at leastsome of the plurality of chambers coupled to a plurality of inlets inthe carrier. The system includes an interface plate adapted to engage atleast one of the inlets in the carrier, a fluid source coupled to theinterface plate and adapted to provide pressurized fluid to at least oneof the inlets in the carrier, and a controller coupled to the fluidsource and to the interface plate for directing fluid from the fluidsource to the carrier.

In other embodiments, the microfluidic device further comprises aplurality of valve lines, and the fluid is directed into at least someof the valve lines by the controller; the controller is further adaptedto open and close at least some of the valve lines; the carrier furthercomprises a plurality of wells, and wherein at least some of the wellsare coupled to corresponding inlets of the plurality of inlets, thecorresponding inlets being adapted to receive a fluid for analysis inthe microfluidic device; the controller is adapted to apply a pressurethrough the interface plate to at least some of the plurality of wellsin order to drive the fluid therein into at least some of the pluralityof chambers; the interface plate comprises two or more separateinterface plates each adapted to engage at least one inlet in thecarrier; the carrier comprises an accumulator chamber having anaccumulator port, and wherein the interface plate comprises a port thatis in fluid communication with the accumulator chamber; the accumulatorchamber further comprises a valve for controlling fluid movement intothe accumulator chamber through the accumulator port, the valve being influid communication with the accumulator port; the valve permits fluidflow into the accumulator chamber through the accumulator port whilerestricting fluid flow out of the accumulator chamber through theaccumulator port; the valve permits fluid flow out of the accumulatorwhen the valve is actuated; the valve is actuated mechanically; thevalve is a check valve; the interface plate comprises a valve actuatorwhich is adapted to engage the valve when the interface plate andcarrier are coupled; the accumulator chamber further comprises a liquid;the accumulator chamber further comprises a gas, or a gas and a liquid;the gas is pressurized relative to a gas pressure outside of theaccumulator chamber; the interface plate further comprises a sealinggasket; the accumulator is adapted to maintain a pressure above adesired pressure level in order to a maintain a valve in themicrofluidic device in a closed state; and the closed valve lasts for atleast two (2) days.

The present invention further provides methods for conducting a step ina protein crystallization condition screening. In one embodiment, themethod includes providing a microfluidic device and performing one ofthe steps from the group consisting of: robotically filling a well inthe microfluidic device with a reagent, robotically moving themicrofluidic device from a robotic liquid dispensing station to adifferent location, robotically placing the microfluidic device into theapparatus; removing the microfluidic device from the apparatus,robotically placing the microfluidic device into an optical inspectionstation, and optically interrogating the microfluidic device using anautomated imaging system. Robotically means movement of the microfluidicdevice caused by a mechanical device under control of a computer orelectronic controller.

The invention provides methods for crystallizing a protein. In oneembodiment the method includes providing a microfluidic device having afirst chamber having a dimension between 1000 μm and 1 μm, a secondchamber having a dimension between 1000 μm and 1 μm, and a channelhaving a dimension between 1000 μm and 1 μm. The first and secondchambers are in fluid communication with each other through the channel.A valve is disposed along the channel which, when actuated to open orclose, controls fluid communication between the first and secondchambers. The method includes introducing a crystallization reagent intothe first chamber, introducing the protein in a solution into the secondchamber, opening the valve so that the solution containing the proteinin the second chamber becomes in fluid communication with thecrystallization reagent in the first chamber, and closing the valveafter a period of time to interrupt fluid communication between thefirst and second chambers.

In some embodiments, the method includes wherein the valve is under thecontrol of an automated valve actuating device; the automated valveactuating device is further under computer control; the valve is openedand closed two or more times; the microfluidic device is a multilayermicrofluidic device; the multilayer microfluidic device comprises atleast one elastomeric layer and the valve is comprises a deflectablemembrane; the two layers of the multilayer microfluidic device comprisean elastomeric material and may be bonded together to form anelastomeric block; the two or more layers of the multilayer microfluidicdevice comprise a first channel in a first layer, and a second channelin a second layer, wherein a portion of the first channel and a portionof the second channel overlap at an overlap region; the first and secondchannels are in fluid communication through a via located at the overlapregion; the overlap region further comprises a deflectable membranedeflectable into either of the first or second channel to control fluidmovement along the first or second channel; and the deflectable membraneis integral to either of the first or second layer.

The invention provides, in one aspect, for a microfluidic device,comprising: a first elastomeric layer having a recess with a widthdimension between 0.1 μm and 1000 μm, a second elastomeric layer havinga recess with a width dimension between 0.1 μm and 1000 μm, and a topsurface, wherein the first elastomeric layer is bonded to the topsurface of the second elastomeric layer to form an elastomeric blockhaving a deflectable portion therein, the elastomeric block having abottom surface defining a surface area, and the elastomeric block havinga height, a substrate having a recess therein and a first surface, thesubstrate having a port in the first surface of the substrate, the portbeing in fluid communication with the recess of the substrate, whereinthe elastomeric block is attached to the substrate to form themicrofluidic device without the elastomeric block occluding the port.

In some embodiments, the port is a well having an opening in the firstsurface of the substrate, the elastomeric block not occluding the wellopening when attached to the substrate, the substrate further comprisesa second surface different than the first surface of the substrate, andwherein the elastomeric block is attached to the second surface of thesubstrate, the first surface is a top surface of the substrate and thesecond surface is a bottom surface of the substrate, the elastomericblock is attached to the first surface of the substrate without theelastomeric block occluding the port, the port is a well, the well has awall having a height that extends above the first surface of thesubstrate where the elastomeric block is attached to the substrate, thewell wall height is coextensive with the elastomeric block height, thewell wall height is less than the elastomeric block height, the wellwall height is greater that the elastomeric block height, the recess isa plurality of recesses and the port is a plurality of ports, whereineach port is in fluid communication with at least one of the pluralityof recesses of the substrate, at least one of the plurality of ports isa well, the well defines a volume between 0.1 μl and 400 μl, the welldefines a volume between 0.1 μl and 250 μl, the well defines a volumebetween 0.1 μl and 100 μl, the well defines a volume between 0.1 ul and10 ul, at least one recess of the plurality of recesses of the substratehas a at least one region having a cross-sectional dimension between 0.1μm and 1000 μm, at least one of the plurality of recesses of thesubstrate has a at least one region having a cross-sectional dimensionbetween 0.1 μm and 500 μm, the recesses of the substrate has a at leastone region having a cross-sectional dimension between 0.1 μm and 100 μm,at least one of the plurality of recesses of the substrate has across-sectional dimension between 0.1 μm and 10 μm, and/or where thesubstrate comprises a polymer, the substrate comprises a polymer isselected from the group consisting of polymethylmethacrylate,polystyrene, polypropylene, polyester, fluoropolymers,polytetrafluoroethylene, polycarbonate, polysilicon, andpolydimethylsiloxane, the substrate comprises glass or quartz, thesubstrate further comprises a sealing layer attached to the substratefor sealing the recesses to form a channel from the recess, the sealinglayer comprises a film, the film is attached by an adhesive, the film isan adhesive film having adhesive thereon prior to attachment of the filmto the substrate, the elastomeric block further comprises a via, the viaprovides fluid communication between the recess in the substrate and therecess in the first elastomeric layer, the via was formed by coring theelastomeric block, the via was formed by drilling the elastomeric block,the via was formed by ablation, the ablation was achieved using a laserbeam, the laser beam was generated by an excimer laser, the via wasformed by etching one of the first or second elastomeric layers, the viais formed one of the first or second elastomeric layers prior to formingthe elastomeric block, the recess in the elastomeric layer overlaps therecesses of the second elastomeric layer, wherein the deflectableportion of the elastomeric block is formed from the second elastomericlayer where the recess of the second elastomeric layer is overlapped bythe recess of the first elastomeric layer to form a deflectable membraneseparating the recesses of the first elastomeric layer from the recessof the second elastomeric layer, the recess of the substrate and the viaand the recess in the first elastomeric layer contain a fluid, thefluid, when at a pressure different than a pressure of a second fluid inthe recess of the second layer, actuates the deflectable membranecausing the deflectable membrane to deflect into one of the recess offirst elastomeric layer or the recesses of the second elastomeric layer,the via is formed by a process using a robotic device movable in x and ydimensions, the robotic device comprises an x,y movable stage, at leastone of the first and second elastomeric layers comprises an elastomericmaterial having a Young's modulus between 1000 Pa and 1,000,000 Pa, atleast one of the first and second elastomeric layers comprises anelastomeric material having a Young's modulus between 10,000 Pa and1,000,000 Pa, at least one of the first and second elastomeric layerscomprises an elastomeric material having a Young's modulus between100,000 Pa and 1,000,000 Pa, at least one of the first and secondelastomeric layers comprises an elastomeric material having a Young'smodulus between 360,000 Pa and 870,000 Pa, at least one of theelastomeric layers comprises polydimethylsiloxane, at least one of theelastomeric layers comprises a polymer made from a two-part polymerforming material, at least one of the elastomeric layers has been plasmaetched, the elastomeric block contacts the substrate, the elastomericblock is bonded to the substrate, between the elastomeric block and thesubstrate further comprises a gasket, the elastomeric block is glued tothe substrate, the port is in fluid communication with an accumulatorchamber, the accumulator chamber has an accumulator port for introducingfluid into the accumulator chamber, the accumulator chamber furthercomprises a valve for controlling fluid movement into the accumulatorchamber through the accumulator port, the valve being in fluidcommunication with the accumulator port, the valve permits fluid flowinto the accumulator chamber through the accumulator port whilerestricting fluid flow out of the accumulator chamber through theaccumulator port, the valve permits fluid flow out of the accumulatorwhen the valve is actuated, the valve is actuated mechanically, thevalve is a check valve, the accumulator further comprises a liquid, theaccumulator chamber further comprises a gas, the accumulator furthercomprises a gas and a liquid, the gas is pressurized relative to a gaspressure outside of the accumulator chamber, the port is a plurality ofports, and the recess in the substrate is a plurality of recesses in thesubstrate, each of the plurality of ports being in fluid communicationwith at least one of the plurality of recesses, each of the plurality ofports are in fluidic communication with one of a plurality of wells, thewells each have an opening in the first surface, the elastomeric blocknot occluding the well opening when attached to the substrate, the wellopenings have a center point, and the plurality of wells is spatiallyarranged such that the center-to-center spacing of each well is that ofthe center-point spacing of a microtiter plate having a format selectedfrom the group of a 96 well microtiter plate, a 384 well microtiterplate, a 864 well microtiter plate, a 1536 well microtiter plate, and a6144 well microtiter plate, the well openings have a center point, andthe plurality of wells is spatially arranged such that the centerpoint-to-center point spacing is about 4.5 mm.

Another aspect of the invention provides for a microfluidic devicecomprising: a first layer having therein a first recess; a second layerhaving a second layer top surface and a second recess therein; asubstrate layer having a top surface, wherein the first layer is bondedto the second layer such that a first channel is formed from the firstrecess and the second layer top surface, and the second layer is bondedto the substrate such that a second channel is formed from the secondrecess and the substrate top surface, and a portion of the first channeloverlaps a portion of the second channel to form a channel overlap; and,a first channel-second channel via establishing fluid communicationbetween the second channel and the first channel at the channel overlap,wherein the first channel-second channel via is formed after the firstlayer and the second layer are bonded together to form a microfluidicblock.

In other aspects, the first channel-second channel via extends from thesecond channel and through and beyond the first channel; the firstchannel-second channel via is formed by laser ablation; at least one orat least two of the layers comprises an elastomer; the substratecomprises a polymer, glass, or quartz; the polymer is selected from thegroup consisting of polymethylmethacrylate, polystyrene, polypropylene,polycarbonate, polysilicon, and plastic; the second layer furthercomprises a third channel formed from a third recess in the second layerand the top surface of the substrate wherein a portion of the thirdchannel and a second portion of the first channel overlap to form asecond overlap and wherein the third channel and the second channel arein fluid communication through a first channel-third channel via locatedat the second overlap; the first channel-second channel via is formedafter the first layer and second layer are bonded; the substrate furthercomprises a substrate recess, a portion of which is overlapped by aportion of the first channel to form a first channel-substrate channeloverlap; a sealing layer having a top surface bonded to the substratesuch that at least one of the substrate recesses forms a substratechannel; and a first channel-substrate channel via located at the firstchannel-substrate channel overlap, wherein the first channel and thesubstrate channel are in fluid communication through the first channel.

Another aspect of the invention provides for increasing the density ofreactions within a microfluidic device by interconnecting channelslocated within different layers of the microfluidic device, wherein saidinterconnections are made using vias, preferably vias formed after twoor more layers containing channels are bonded together, more preferablyby forming the vias using a laser ablation tool.

The invention provides, in one aspect, for a carrier for holding amicrofluidic device comprising: a housing, the housing defining achamber therein and having a receiving portion for receiving themicrofluidic device; a connection block for retaining the microfluidicdevice, wherein the connection block is attachable to the microfluidicdevice through one or more prongs, and the microfluidic device, whenretained by the connection block, is insertable into the receivingportion of the housing.

Other embodiments include having the one or more prongs be two or moreprongs, having at least one of the one or more prongs is a tube, havingthe receiver has at least one slot for guiding and retaining themicrofluidic device when inserted into the receiving portion, having thereceiver further comprises one or more pipette supports for guiding apipette tip into the microfluidic device when inserted into thereceiving portion, including one or more accumulators for providingfluid under pressure to the microfluidic device when inserted into thereceiving portion, preferably where at least one accumulator furthercomprises a check valve, having the housing comprises a housing base anda housing cover, preferably where an accumulator is attached to thehousing, and preferably where the housing cover and the housing base aresealed together by a gasket, including a humidity control materialwithin the housing for providing humidity control, preferably where thehumidity control material is selected from the group consisting of asponge, a gel matrix, a desiccant, and a woven material, having thehousing is preferably be made from a polymer, more preferably where thepolymer is either polycarbonate or acrylic or polystyrene, preferablywhere the accumulator is in fluid communication with the connectionblock through one or more accumulator-connection block tubes, whereinthe accumulator-connection block tubes are preferably flexible, having afirst tube of the one or more tubes is in communication with themicrofluidic device for controlling one or more first valves, preferablywherein a second tube of the one or more tubes is in communication withthe microfluidic device for controlling one or more second valves, forexample, but not limited to, wherein the first valves are interfacevalves and/or wherein the second valves are containment valves.

In another embodiment, the present invention provides a device forpositioning protein crystal within an energy beam comprising a chip forholding the crystal therein, the chip being made from an elastomericblock having disposed therein a deflectable membrane. The deviceincludes an adapter plate for connecting the chip to a post, the chipbeing connected to the adapter plate through one or more postspenetrating into the chip, and a goniometer, wherein the post isconnected to the post for positioning the crystal within the beam. Inother aspects, the adapter plate is movably translatable so as tofurther position the crystal within an axis perpendicular to the beam;and the goniometer is rotatable about an axis perpendicular andintersecting the beam, and the chip is rotated about the axis of thebeam so as to expose different facets of the crystal to the beam.

Embodiments of the invention are directed to a method of conducting areaction at a selected temperature or range of temperatures over time.An array device is provided. The array device contains a plurality ofseparate reaction chambers and comprises an elastomeric block formedfrom a plurality of layers. At least one layer has at least one recessformed therein. The recess has at least one deflectable membraneintegral to the layer with the recess. The array device furthercomprises a thermal transfer device proximal to at least one of thereaction chambers. The thermal transfer device is formed to contact athermal control source. Reagents for carrying out a desired reaction areintroduced into the array device. The array device is contacted with athermal control device such that the thermal control device is inthermal communication with the thermal control source so that atemperature of the reaction in at least one of the reaction chamber ischanged as a result of a change in temperature of the thermal controlsource.

In different embodiments, the thermal transfer device may comprise asemiconductor, such as silicon, may comprise a reflective material,and/or may comprise a metal.

The thermal control device may be adapted to apply a force to thethermal transfer device to urge the thermal transfer device towards thethermal control source. The force may comprise a magnetic,electrostatic, or vacuum force in different embodiments. For example, inone embodiment, the force comprises a vacuum force applied towards thethermal transfer device through channels formed in a surface of thethermal control device or the thermal transfer device. A level of vacuumachieved between the surface of the thermal control device and a surfaceof the thermal transfer device may be detected. Such detection may beperformed with a vacuum level detector located at a position along thechannel or channels distal from a location of a source of vacuum. Whenthe vacuum does not exceed a preset level, an alert may be manifested ora realignment protocol may be engaged.

The array device may be contacted with the thermal control device byemployment of one or more mechanical or electromechanical positioningdevices. Carrying out of the method may be automatically controlled andmonitored. For example, such automatic control and monitoring may beperformed with an automatic control system in operable communicationwith a robotic control system for introducing and removing the arraydevice from the thermal control device. The progress of the reactionsmay also be monitored.

A device may be provided comprising the array device. A unit may beprovided comprising the thermal control device. A system may be providedcomprising the array device and the thermal control device.

In other embodiments, a microfluidic system is provided. An array deviceis provided for containing a plurality of separate reaction chambersdisposed within a reaction area and in fluid communication with fluidinlets to the array device disposed outside the reaction area. The arraydevice comprises an elastomeric block formed from a plurality of layers.At least one layer has at least one recess formed therein. The recesshas at least one deflectable membrane integral to the layer with therecess. A carrier is adapted to hold the array device and has aplurality of fluid channels interfaced with the fluid inlets. A thermaltransfer interface comprises a thermally conductive material disposed toprovide substantially homogeneous thermal communication from a thermalcontrol source to the reaction area.

In different embodiments, the thermally conductive material may bereflective, may comprise a semiconductor such as silicon or polishedsilicon, and/or may comprise a metal.

In one embodiment, the reaction area is located within a central portionof the array device and the fluid inlets are disposed at a periphery ofthe array device. The array device may be coupled with the carrier atthe periphery of the array device and the thermally conductive materialmay be coupled with a surface of the array device at the reaction area.

In some embodiments, a means is provided for applying a force to thethermal transfer interface to urge the thermal transfer interfacetowards the thermal control source. The means for applying the force maycomprise a means for applying a vacuum source towards the thermaltransfer interface through channels formed in a surface of a thermalcontrol device or in the thermal transfer device. A vacuum leveldetector may be provide for detecting a level of vacuum achieved betweenthe surface of the thermal control device and a surface of the thermaltransfer device. In one embodiment, the vacuum level detector is locatedat a position along the channel or channels distal from a location of asource of vacuum.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are simplified cross-sections of prior art elastomericblocks;

FIG. 2 is a an exploded view of a prior art carrier and microfluidicdevice;

FIG. 3 is an exploded view of a carrier and microfluidic deviceaccording to an embodiment of the present invention;

FIG. 4 depicts a perspective view of a carrier according to anembodiment of the present invention;

FIG. 5 depicts a plan view of the carrier shown in FIGS. 3 and 4;

FIG. 6 depicts a cross-sectional view of the accumulator chamber of thecarrier shown in FIGS. 3-5;

FIG. 7 is a perspective view of another carrier according to anembodiment of the present invention;

FIG. 8A depicts a substrate of a microfluidic device that has integratedpressure accumulator wells according to an embodiment of the presentinvention;

FIG. 8B depicts an exploded view of the microfluidic device shown inFIG. 8A, and further including an elastomeric block;

FIG. 8C is an overall view of the microfluidic device shown in FIG. 8B;

FIG. 8D is a plan view of the microfluidic device shown in FIG. 8B;

FIG. 8E depicts a plan view of the microfluidic device shown in FIG. 8B;

FIG. 8F depicts a bottom plan view of the microfluidic device shown inFIG. 8B;

FIG. 8G depicts a cross-sectional view of the microfluidic device shownin FIG. 8B;

FIGS. 9A and 9B are close-up views of a fluidic interface according toan embodiment of the present invention;

FIG. 9C is a cross sectional view of a via for use in some embodimentsof microfluidic devices of the present invention;

FIG. 9D is a blown up view of a via for use in some embodiments ofmicrofluidic devices of the present invention;

FIG. 10 is a plan view of one embodiment of a chip for use with thepresent invention;

FIG. 11A-D are close up plan view of exemplary metering cells in variousvalve states according to embodiments of the present invention;

FIG. 11E is a photograph of an exemplary metering cell format;

FIG. 11F depicts a high density formal for reacting a plurality ofsamples according to an embodiment of the present invention;

FIG. 11G is a plan view of one embodiment of a chip for use with thepresent invention;

FIG. 12A is a perspective view of a station for actuating a microfluidicdevice according to an embodiment of the present invention;

FIGS. 12B and 12D are perspective and side views, respectively, of thestation of FIG. 12A with the platen in a down position;

FIG. 12C is a side view of the station of FIG. 12A with the platen in anup position;

FIG. 12E depicts a close-up view of the platen of FIG. 12A;

FIG. 12F depicts a cut-away side view of the platen of FIG. 12A;

FIG. 12G is a close-up view of a purge actuator acting on a check valveaccording to an embodiment of the present invention;

FIG. 12H depicts a cut-away view of a platen urged against the upperface of a microfluidic device according to an embodiment of the presentinvention;

FIG. 13 is a rear plan view of fluidic routing within a plate interfaceor platen according to an embodiment of the present invention;

FIG. 14A is perspective view of a carrier in accordance with anembodiment of the present invention;

FIG. 14B is a top view of an integrated carrier and chip according to anembodiment of the present invention;

FIG. 15A is a simplified overall view of a system according to anembodiment of the present invention;

FIG. 15B is a perspective view of a receiving station in the system ofFIG. 15A;

FIG. 15C is a rear plan view of fluidic routing within a plate interfaceor platen according to another embodiment of the present invention;

FIGS. 16A and 16B are cross sectional side views showing an interfaceplate mated to a carrier according to an embodiment of the presentinvention;

FIG. 17 is an example screen shot available with the system of FIG. 15A;

FIG. 18A is a perspective view of an integrated carrier according to anembodiment of the present invention;

FIG. 18B is a perspective view of an integrated carrier according toanother embodiment of the present invention for use with PCR;

FIG. 19A is a simplified cross sectional view of a system for retainingthe carrier of FIG. 18A;

FIG. 19B is a simplified cross sectional view of a system for retainingthe carrier of FIG. 18B;

FIG. 19C is a simplified plan view of a portion of the carrier of FIG.18B;

FIG. 19D is a simplified plan view of a vacuum chuck for use with thesystem of FIG. 19B; and

FIG. 20 is a simplified overall view of a vacuum chuck for use with thesystem of FIG. 18B.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Systems of the present invention will be particularly useful formetering small volumes of material in the context of performingcrystallization of target material. A host of parameters can be variedduring such crystallization screening. Such parameters include but arenot limited to: 1) volume of crystallization trial, 2) ratio of targetsolution to crystallization solution, 3) target concentration, 4)cocrystallization of the target with a secondary small or macromolecule,5) hydration, 6) incubation time, 7) temperature, 8) pressure, 9)contact surfaces, 10) modifications to target molecules, 11) gravity,and (12) chemical variability. Volumes of crystallization trials can beof any conceivable value, from the picoliter to milliliter range.

The length of time for crystallization experiments can range fromminutes or hours to weeks or months. Most experiments on biologicalsystems typically show results within 24 hours to 2 weeks. This regimeof incubation time can be accommodated by the microfluidics devices inaccordance with embodiments of the present invention.

The temperature of a crystallization experiment can have a great impacton success or failure rates. This is particularly true for biologicalsamples, where temperatures of crystallization experiments can rangefrom 0-42° C. Some of the most common crystallization temperatures are:0, 1, 2, 4, 5, 8, 10, 12, 15, 18, 20, 22, 25, 30, 35, 37, and 42.Microfluidics devices in accordance with embodiments of the presentinvention can be stored at the temperatures listed, or alternatively maybe placed into thermal contact with small temperature control structuressuch as resistive heaters or Peltier cooling structures. In addition,the small footprint and rapid setup time of embodiments in accordancewith the present invention allow faster equilibration to desired targettemperatures and storage in smaller incubators at a range oftemperatures.

Embodiments of microfluidic structures in accordance with the presentinvention may be employed for applications other than crystallizationscreening. Examples of such applications include those described inInternational Application No. PCT/US01/44869, filed Nov. 16, 2001,entitled “Cell Assays And High Throughput Screening,” herebyincorporated by reference for all purposes. Examples of microfluidicstructures suitable for performing such applications include thosedescribed herein, as well as others described in U.S. patent applicationSer. No. 10/118,466, filed Apr. 5, 2002, entitled “Nucleic AcidAmplification Utilizing Microfluidic Devices,” the complete disclosureof which is hereby incorporated by reference for all purposes.

An embodiment of a method of fabricating a microfluidic device inaccordance with the present invention comprises etching a top surface ofa glass substrate to produce a plurality of wells, molding an elastomerblock such that a bottom surface bears a patterned recess, placing abottom surface of the molded elastomer block into contact with the topsurface of the glass substrate, such that the patterned recess isaligned with the wells to form a flow channel between the wells.

An embodiment of a method for forming crystals of a target materialcomprises priming a first chamber of an elastomeric microfluidic devicewith a first predetermined volume of a target material solution. Asecond chamber of an elastomer microfluidic device is primed with asecond predetermined volume of a crystallizing agent. The first chamberis placed into fluidic contact with the second chamber to allowdiffusion between the target material and the crystallizing agent, suchthat an environment of the target material is changed to cause formationof crystal.

In yet another aspect, chambers or metering cells may be formed in afirst elastomer layer, said chambers or metering cells being in fluidcommunication through fluid channels, and a second layer having formedtherein control channels, wherein deflectable membranes between thefirst and second layers are deflectable into the first layer to controlfluid flow through the fluid channels. A substrate may be mated to thefirst and second layers to impart rigidity or provide for additionalfluidic interconnections. The microfluidic devices then may be used inconjunction with carriers and/or systems for providing process controlas further detailed herein.

The present invention provides for microfluidic devices and methods fortheir use. The invention further provides for apparatus for using themicrofluidic devices of the invention, analyze reactions carried out inthe microfluidic devices, and systems to generate, store, organize, andanalyze data generated from using the microfluidic devices. Devices,systems and methods of the present invention will be particularly usefulwith various microfluidic devices, including without limitation theTopaz® series of devices available from Fluidigm, Corporation of SouthSan Francisco, Calif. The present invention also will be useful forother microfabricated fluidic devices utilizing elastomer materials,including those described generally in U.S. patent application Ser. No.09/826,583, filed Apr. 6, 2001, entitled “Microfabricated ElastomericValve And Pump Systems,” Ser. No. 09/724,784, filed Nov. 28, 2000,entitled “Microfabricated Elastomeric Valve And Pump Systems,” and Ser.No. 09/605,520, filed Jun. 27, 2000. entitled “MicrofabricatedElastomeric Valve And Pump Systems.” These patent applications arehereby incorporated by reference.

High throughput screening of crystallization of a target material, orpurification of small samples of target material by recrystallization,is accomplished by simultaneously introducing a solution of the targetmaterial at known concentrations into a plurality of chambers of amicrofabricated fluidic device. The microfabricated fluidic device isthen manipulated to vary solution conditions in the chambers, therebysimultaneously providing a large number of crystallization environments.Control over changed solvent conditions may result from a variety oftechniques, including but not limited to metering of volumes of acrystallizing agent into the chamber by volume exclusion, by entrapmentof liquid volumes determined by the dimensions of the microfabricatedstructure, or by cross-channel injection into a matrix of junctionsdefined by intersecting orthogonal flow channels.

Crystals resulting from crystallization in accordance with embodimentsof the present invention can be utilized for x-ray crystallography todetermine three-dimensional molecular structure. Alternatively, wherehigh throughput screening in accordance with embodiments of the presentinvention does not produce crystals of sufficient size for direct x-raycrystallography, the crystals can be utilized as seed crystals forfurther crystallization experiments. Promising screening results canalso be utilized as a basis for further screening focusing on a narrowerspectrum of crystallization conditions, in a manner analogous to the useof standardized sparse matrix techniques.

Systems and methods in accordance with embodiments of the presentinvention are particularly suited to crystallizing larger biologicalmacromolecules or aggregates thereof, such as proteins, nucleic acids,viruses, and protein/ligand complexes. However, crystallization inaccordance with the present invention is not limited to any particulartype of target material. Further, while embodiments of the presentinvention discussed utilize diffusion of crystallizing agent in theliquid phase, vapor diffusion is another technique that has beenemployed to induce crystal formation.

Embodiments of microfluidic devices in accordance with the presentinvention may utilize on-chip reservoirs or wells. However, in amicrofluidic device requiring the loading of a large number ofsolutions, the use of a corresponding large number of input tubes withseparate pins for interfacing each well may be impractical given therelatively small dimensions of the fluidic device. In addition, theautomated use of pipettes for dispensing small volumes of liquid isknown, and thus it therefore may prove easiest to utilize suchtechniques to pipette solutions directly on to wells present on the faceof a chip.

Capillary action may not be sufficient to draw solutions from on-chipwells into active regions of the chip, particularly where dead-endedchambers are to be primed with material. In such embodiments, one way ofloading materials into the chip is through the use of externalpressurization. Again however, the small dimensions of the devicecoupled with a large number of possible material sources may renderimpractical the application of pressure to individual wells through pinsor tubing.

Turning now to FIG. 3, a microfluidic device according to an embodimentof the present invention will be described having one or more integratedfluid pressure storage chambers or accumulators to provide a source offluid pressure to one or more deflectable membranes within themicrofluidic device. FIG. 3 depicts a preferred embodiment of a carrier323 with an integrated pressure accumulator. Carrier 323 comprises acarrier base 301 which has a receiving area 300 for receiving andmaintaining the position of a microfluidic device 305 inside carrier323. Microfluidic device 305 may be a wide range of devices within thescope of the present invention, including Topaz® 1.96 and Topaz® 4.96chips available from Fluidigm Corporation.

Microfluidic device 305 comprises one or more well rows 306 having oneor more inlet wells 307 that are in fluid communication with channelsinside microfluidic device 305, a containment valve inlet 320, aninterface valve inlet 321, and a sample inlet 324. A carrier top 309includes pressure cavities 310 and 311 which are positioned in contactwith well rows 306 to form a common pressure chamber over each well 307for each well row 306. Pressure chamber inlets 313 and 314 are used tosupply gas pressure to each pressure chamber when formed with eachpressure cavity contacting the surface of microfluidic device 305.

Carrier 323 further includes a pressure accumulator 324 which ispreferably formed by attaching an accumulator top portion 303 to aportion of carrier base 301 forming an accumulator chamber 304 therein.Fluid, preferably gas, is introduced into accumulator chamber 304through an accumulator inlet 317 which is in fluid communication withaccumulator chamber 304. Preferably, an accumulator check valve 302 isplaced in-line between accumulator inlet 317 and accumulator chamber 304to maintain fluid pressure within accumulator chamber 304 even after thedisconnection of a fluid pressure source (not shown) from accumulatorinlet 317. Preferably, accumulator check valve 302 is housed in a“dry-well” inside of accumulator chamber 304 when gas is used topressurize accumulator chamber 304 while a portion of accumulatorchamber 304 contains a liquid to create hydraulic pressure with theliquid contained therein. The liquid, under hydraulic pressure, can bein turn used to actuate a deflectable portion, such as a membrane,preferably a valve membrane, inside of microfluidic device 305 bysupplying hydraulic pressure through an accumulator outlet 316 that isin fluid communication with accumulator chamber 304 and at least onechannel within microfluidic device 305.

In the embodiment shown in FIG. 3, carrier top 309 is attached tocarrier base 301 by one or more screws 309 being threaded intocorresponding one or more screw holes 333 of carrier base 301 so that acompressive force is maintained between carrier top 309 and the topsurface of microfluidic device 305 so that pressure cavities 310 and 311form fluid tight seals around well rows 306. An interface pressuresupply line inlet 318 connects to an interface pressure supply line 319which is also inserted into interface valve inlet 321 of microfluidicdevice 305 to provide a source of pressurized fluid, preferably gas, orhydraulic pressure to a second channel within microfluidic device 305 toactuate at least one second deflectable portion, preferably adeflectable membrane of a second interface valve, within microfluidicdevice 305. One or more metering cells 308 within microfluidic device305 are in fluid communication with well inlets 307 and a sample inlet334. In some embodiments, a protein crystallization metering cell, suchas one described in Hansen, is provided, wherein a first and a secondchamber are in fluid communication through one or more interfacechannels therebetween, wherein the interface channels further comprisean interface valve for controlling diffusion or fluid movement betweeneach chamber. Each chamber is further in fluid communication with aninlet for introducing a fluid into each chamber, the inlets being influid communication with the chambers through channels inside themicrofluidic device.

One method of using carrier 323 according to the present invention willbe described. With carrier top 309 off, wells 307 are filled withreagents. A sample solution is injected into sample inlet 334 using amicropipettor. The interface valve within each metering cell 308 isclosed by applying pressure to interface valve inlet 321 throughinterface pressure supply line 319. The sample solution may be furthermoved inside of microfluidic device 305 by further applying pressure(e.g., in the form of gas pressure) into sample inlet 334 to push thesample solution into the sample reagent of metering cell 308. Hydraulicliquid, preferably water, more preferably oil, still more preferablyKrytox(R) GL100(tm) oil, which is polyhexafluoropropylene oxide, or ablend of oils and other solvents, such as water, is introduced intointerface valve inlet 320 and containment valve inlet 321, preferably byusing a micropipettor. Containment line 300 and control line 319 areinserted into inlets 320 and 321, respectively, and carrier top 309 isaffixed to carrier base 301 with microfluidic device 305 therebetween.

FIG. 4 depicts a perspective view of carrier 323 shown in FIG. 3. FIG. 5depicts a plan view of the carrier shown in FIGS. 3 and 4. FIG. 6depicts a cross-sectional view of accumulator chamber 304 insideaccumulator 324 showing an angled chamber floor angled downward withrespect to accumulator chamber cover 303 which permits liquid to draintowards line 300, and also shows access screw 335 which can be removedfor adding or removing fluids, preferably liquids as shown partiallyoccupying accumulator chamber 304. A side view of check valve 302 isshown situated inside of dry-well 340 defined by dry-well wall(s) 340.1.

FIG. 7 depicts a carrier similar to the carrier shown in FIGS. 3-6,however, instead of a single accumulator being present, two separateaccumulators 303.1 and 303.2 are integrated into the carrier. In apreferred use, the second accumulator is used to actuate, and maintainactuation of a second deflectable portion of the microfluidic device,preferably a second deflectable membrane valve. In a particularlypreferred embodiment, the first accumulator is used to actuate interfacevalves within a metering cell, and the second accumulator is used toactuate containment valves within a metering cell, independent of eachother. In yet other embodiments, a plurality of accumulators may also beincluded to provide for independent actuation of additional valvesystems or to drive fluid through a microfluidic device.

In an alternative embodiment of the present invention, FIG. 8A depicts asubstrate 800 of a microfluidic device that has integrated pressureaccumulator wells 801 and 802, each having therein a drywell 803, 804for receiving a valve, preferably a check valve attached to a cover (seeFIG. 8B). Substrate 800 further includes one or more well banks 806 a,b, c, and d, each having one or more wells 805 located therein. Each ofthe wells 805 of substrate 800 have channels leading from well 805 toelastomeric block location 807 within substrate 800 for attaching anelastomeric block, preferably an elastomeric block formed from two ormore layers of elastomeric material having microfabricated recesses orchannels formed therein.

FIG. 8B depicts an exploded view of a complete microfluidic device 899comprising the components shown in FIG. 8A, and further comprising anelastomeric block 808 which is attached, or more preferably bonded, andyet more preferably directly bonded, preferably without use of adhesivesto the elastomeric block location 807 of substrate 800 to form thecomplete microfluidic device 899 (FIG. 8C). Within elastomeric block 808are one or more channels in fluid communication with one or more vias814, which in turn provide fluid communication between the channelswithin the elastomeric block and channels within the substrate whichthen lead to wells 805 within well rows 806 a-d to provide for fluidcommunication between wells 805 of substrate 800 and the channels withinelastomeric block 808. Accumulator well tops 809 and 810 are attached toaccumulator wells 801 and 802 to form accumulator chambers 815 and 816.Accumulator well tops 809 and 810 include valves 812 and 811,respectively, which are preferably check valves for introducing andholding gas under pressure into accumulator chambers 815 and 816. Valves811 and 812 are situated inside of drywells 802 and 804 to keep liquid,when present in accumulator chambers 815 and 816, from contacting valves811 and 812. Valves 811 and 812 preferably may be mechanically opened bypressing a shave, pin or the like, within a preferred check valve toovercome the self closing force of the check valve to permit release ofpressure from the accumulator chamber to reduce the pressure of thefluid contained within the accumulator chamber.

FIG. 8D depicts a plan view of microfluidic device 899 and wells 805,wherein a port is located adjacent the base of the well, preferably thebottom, or alternatively the side of well 805 for passage of fluid fromthe well into a channel formed in substrate 800, preferably on the sideof substrate 800 opposite of well 805. In a particularly preferredembodiment, substrate 800 is molded with recesses therein, the recessesbeing made into channels by a sealing layer, preferably an adhesive filmor a sealing layer.

Substrate 800 and its associated components may be fabricated frompolymers, such as polypropylene, polyethylene, polycarbonate,high-density polyethylene, polytetrafluoroethylene PTFE or Teflon (R),glass, quartz, or a metal (for example, aluminum), transparentmaterials, polysilicon, or the like. Accumulator well tops 809 and 810further may comprise access screws 812 which can be removed to introduceor remove gas or liquid from accumulator chambers 815 and 816.Preferably, valves 812 and 811 can be actuated to release fluid pressureotherwise held inside of accumulator chambers 815 and 816. Notch 817 isused to assist correct placement of the microfluidic device into otherinstrumentation, for example, instrumentation used to operate or analyzethe microfluidic device or reactions carried out therein. FIG. 8Dfurther depicts a hydration chamber 850 surrounding elastomeric blockregion 807, which can be covered with a hydration cover 851 to form ahumidification chamber to facilitate the control of humidity around theelastomeric block. Humidity can be increased by adding volatile liquid,for example water, to humidity chamber 851, preferably by wetting ablotting material or sponge. Polyvinyl alcohol may preferably be used.Humidity control can be achieved by varying the ratio of polyvinylalcohol and water, preferably used to wet a blotting material or sponge.Hydration can also be controlled by using a humidity control device suchas a HUMIDIPAK™ humidification package which, for example, uses a watervapor permeable but liquid impermeable envelop to hold a salt solutionhaving a salt concentration suitable for maintaining a desired humiditylevel. See U.S. Pat. No. 6,244,432 by Saari et al, which is hereinincorporated by reference for all purposes including the specificpurpose of humidity control devices and methods. Hydration cover 850 ispreferably transparent so as to not hinder visualization of eventswithin the elastomeric block during use. Likewise, the portion ofsubstrate 800 beneath the elastomeric block region 807 is preferablytransparent, but may also be opaque or reflective.

FIG. 8E depicts a plan view of substrate 800 with its channels formedtherein providing fluid communication between wells 805 and elastomericblock 808 (not shown) which is attached to substrate 800 withinelastomeric block region 807, through channels 872. Accumulator chambers801 and 802 are in fluid communication with elastomeric block region 807and ultimately, elastomeric block 808, through channels 870.

FIG. 8F depicts a bottom plan view of substrate 800. In a particularlypreferred embodiment, recesses are formed in the bottom of substrate 800between a first port 890 which passes through substrate 800 to theopposite side where wells 805 are formed and a second port 892 whichpasses through substrate 800 in fluid communication with a via inelastomeric block 808 (not shown).

FIG. 8G depicts a cross-sectional view of substrate 800 with elastomericblock 808 situated in elastomeric block region 807 along with sealinglayer 881 attached to the side of substrate 800 opposite of elastomericblock 808. Well 805 is in fluid communication with elastomeric block 808through first port 890, channel 870, and second port 892 and into arecess of elastomeric layer 808, which is sealed by a top surface 897 ofsubstrate 800 to form a channel 885. Sealing layer 881 forms channel 870from recesses molded or machined into a bottom surface 898 substrate800. Sealing layer 881 is preferably a transparent material, forexample, polystyrene, polycarbonate, or polypropylene. In oneembodiment, sealing layer 881 is flexible such as in adhesive tape, andmay be attached to substrate 800 by bonding, such as with adhesive orheat sealing, or mechanically attached such as by compression.Preferably materials for sealing layer 881 are compliant to form fluidicseals with each recess to form a fluidic channel with minimal leakage.Sealing layer 881 may further be supported by an additional supportlayer that is rigid (not shown). In another embodiment, sealing layer881 is rigid.

FIG. 9A depicts a close-up detail of the fluidic interface betweenelastomeric block 808 and elastomeric block region 807 of substrate 800.As described in Unger and Hansen, elastomeric blocks may be formed frommultiple layers of elastomeric material bonded together to form anelastomeric block. Preferably at least two of the layers of theelastomeric block have recesses. For example, a first elastomeric layerhaving recesses formed therein is bonded to a second elastomeric layerhaving recesses formed therein to form an elastomeric block havingrecesses formed therein. The recesses of the first elastomeric layer arewholly or partly closed off to form channels in the first elastomericlayer. The recesses formed in the second elastomeric layer are likewisewholly or partly closed off to form channels in the second elastomericlayer when the elastomeric layer is bonded to a substrate, therebyforming a microfluidic device having multiple layers with channelsformed therein.

Turning to FIGS. 9A and 9B, a first elastomeric layer 920 having abottom surface with first recesses 901 formed therein and secondelastomeric layer 923 having a top surface and a bottom surface withsecond recesses 905 formed therein are bonded together to formelastomeric block having channel 907 (formed from first recess 901 andthe top surface of second elastomeric layer 923. Substrate 800 isattached to the bottom surface of the second elastomeric layer 923 toform channel 909 from top surface 897 of substrate 800 and the bottomsurface of second elastomeric layer 923. Port 892 may connect channel872 of substrate 800 with channel 909 of the second elastomeric layer,which is partly formed by the top surface of substrate 800.Alternatively as shown in FIGS. 9A-9B, port 892 connects channel 872 ofsubstrate 800 with channel 907 for first elastomeric layer 920 ofelastomeric block 808 through a via 950. Via 950 is formed about normalto substrate surface 897, preferably formed in second elastomeric layer923, prior to its bonding with elastomeric layer 920, and morepreferably after the first and second elastomeric layers are bondedtogether. See co-pending and commonly assigned U.S. Provisional PatentApplication Ser. No. 60/557,715 by Unger filed on Mar. 29, 2004, whichis incorporated by reference for all purposes and the specific purposeof teaching via formation using automated laser ablation systems andmethods. Exemplary methods for creating vias include microfabricatingwhile forming second elastomeric layer 923, laser drilling, laserdrilling with a CO₂ laser, laser drilling with an excimer laser,drilling mechanically, and coring, preferably wherein the drilling isperformed by a robotic drill system, preferably one having an x,yautomated stage.

FIG. 9B depicts the microfluidic device of FIG. 9A, wherein channel 907of first elastomeric layer 920 overlaps channel 909 of secondelastomeric layer 923 to form a deflectable portion within theelastomeric block, preferably an elastomeric membrane, preferably formedfrom a portion of second elastomeric layer 923. Fluid pressure istransmitted to channel 907 of first elastomeric layer 920 from apressurized fluid source (not shown) through channel 872, port 892, andvia 950 to cause elastomeric membrane 990 to deflect downward to controlfluid flow or diffusion through channel 909 of second elastomeric layer923.

FIG. 9C depicts a cross sectional view of another preferred use of a viain the microfluidic devices described herein. Microfluidic block 921includes first layer 920 having first layer recess (or channel whenbonded to a second layer) 907 formed therein and second layer 923 havingsecond layer recesses (or channels when bonded to a substrate) 950therein. Two second layer channels are in fluid communication through afirst layer channel by way of two or more vias 950. Preferably, at leastone via 950 is in further fluid communication with well 999 of substrate800 through a substrate recess 892 (or channel if a sealing layer (notshown) is bonded to substrate 800). At least one second layer channel909 is overlapped by a portion of first layer channel 907 without beingin fluid communication. In the embodiment shown in FIG. 9C, a higherdensity of reaction and/or detection zones per unit area of microfluidicdevice may be achieved because a fluid channel in one layer can berouted over or under an intervening fluid channel within the same layer.Ablation debris chambers 989 are present to catch debris produced fromlaser ablating via 950. Debris chamber 989 may be cast into layer 920 bytwo-layer casting methods, wherein after a first layer of photoresisthas been patterned and developed, a second layer of photoresist isoverlaid over the first pattern, and a second pattern is formed upon thepattern of the first photoresist layer such that a regions ofphotoresist pattern may be of different heights. Multiple layers can bebuilt up upon one another to create patterns of varying heights.Different photoresist materials may also be used so that, for example,the upper layer of photoresist is capable of reflowing when heated,while the lower layer is made of a photoresist that does notsubstantially reflow at the same heated temperature.

FIG. 9D depicts a blown up view of a via 950 that interconnects channelsfrom two different layers. Microfluidic block 921 is formed from firstlayer 920 having channel 907 therein, and second layer 923 having secondchannel 909 formed therein. Via 950 interconnects channels 907 and 909together. Also shown is debris chamber 989 which was cast into layer 920by a multi-height molding process as described above. When via 950 isformed by laser ablation, debris or material from one of the layers mayreside in the upper portion of channel 907 where the via is formed.Providing a chamber for such debris or material to reside in afterablation helps to prevent closure or stenosis of channel 907 or 909.

The flow channels of the present invention may optionally be designedwith different cross sectional sizes and shapes, offering differentadvantages, depending upon their desired application. For example, thecross sectional shape of the lower flow channel may have a curved uppersurface, either along its entire length or in the region disposed underan upper cross channel). Such a curved upper surface facilitates valvesealing, as follows. Membrane thickness profiles and flow channelcross-sections contemplated by the present invention includerectangular, trapezoidal, circular, ellipsoidal, parabolic, hyperbolic,and polygonal, as well as sections of the above shapes. More complexcross-sectional shapes, such as an embodiment with protrusions or anembodiment having concavities in the flow channel, are also contemplatedby the present invention.

In addition, while the invention is described primarily in conjunctionwith an embodiment wherein the walls and ceiling of the flow channel areformed from elastomer, and the floor of the channel is formed from anunderlying substrate, the present invention is not limited to thisparticular orientation. Walls and floors of channels could also beformed in the underlying substrate, with only the ceiling of the flowchannel constructed from elastomer. This elastomer flow channel ceilingwould project downward into the channel in response to an appliedactuation force, thereby controlling the flow of material through theflow channel. In general, monolithic elastomer structures are preferredfor microfluidic applications. However, it may be useful to employchannels formed in the substrate where such an arrangement providesadvantages. For instance, a substrate including optical waveguides couldbe constructed so that the optical waveguides direct light specificallyto the side of a microfluidic channel.

FIG. 10 depicts a plan view of a preferred embodiment wherein ninety-six(96) separate metering cells are formed within an elastomeric block 808.In a preferred embodiment, hydration lines 1010 are provided adjacenteach elastomeric block inlet which connects ports within substrate 800(not shown) to channels within elastomeric block 808, to provide asource of solutions at a selected osmolarity to provide a source ofhydration and/or osmo-regulation to portions of elastomeric block 808.

FIG. 11A depicts a close-up plan view of an exemplary metering cell usedfor protein crystallization wherein fluid flow in adjacent channels andchambers is controlled by deflectable membrane valves, preferablyopposing “T” or tee shaped interdigitated valves 1100. In preferredembodiments, when a series of channels and reagent chambers are locatedin close proximity such that osmolarity differences between adjacentreagent chambers or channels may cause migration of fluid, typically invapor form, through the elastomeric layers of the elastomeric block,using discontinuous valve lines serve to “osmotically” isolate reagentchambers when compared to linear valve lines 119 which have a shorterfluid distance between each chamber.

FIG. 11B depicts a valve state for a metering cell. Within metering cell1101 show, reagent chambers 1103 and protein chamber 1104 are isolatedfrom each other by the actuation of interface valves 1106 while reagentand protein solution are introduced into each respective chamber. Oncefilled, containment valves 1109 are closed, as shown in FIG. 11C andfree interface diffusion is performed by opening interface valves 1106.As shown in FIG. 11D, diffusion may be interrupted by closing interfacevalve 1106 to permit, for example, dehydration to occur if the ambienthumidity around or within elastomeric block 808 is reduced.

FIG. 11E is a photograph of an exemplary metering cell format.

FIG. 11F depicts a high density format for reacting a plurality ofsamples with a plurality of reagents, for example, preferably four (4)samples with ninety-six (96) reagents; eight (8) samples with ninety-two(92) reagents, and so forth, including, but not limited to forty-eight(48) samples with forty-eight (48) reagents. Each reaction pair may beseparately mixed or combined, such as by diffusion, the format utilizingfluid channel overpasses or underpasses to route other intervening fluidchannels. FIG. 11F is a close up view of an example of a use of vias toincrease the reaction/detection region density of a microfluidic chip. Aclose up view 11110 is provided of four sets of metering cells forcarrying out reactions such as protein crystallization experiments.Metering cell 11100 comprises four sets of chambers in each set having afirst chamber and a second chamber in fluid communication and separatedby an interface valve 11020. With interface valve 11020 closed, reagentsare introduced though ports such as port 11050 which is in fluidcommunication with a metering cell 11100 for filling reagent chambers11030, and sample inlet ports 11080 and 11070, and two of which are notshown, such as protein samples, which are transported to sample chambers11090 through channels which are interconnected through vias 11040,which allow for the samples to pass over the sample branch channels11120. Widened channel paths, such as 11020 a indicate where adeflectable membrane valve is present that is formed by the overlappingof a first layer channel and a second layer channel. Comparativelynarrower channel segments represent, when overlapping other channels,regions where a deflectable membrane is not formed and therefore doesnot act as a valve. The architectures described herein this applicationmay, as one of skill in the art would realize, be reversed in order. Forexample, a fluid layer may be formed inside of a thicker layer, and athinner layer may be used as a control layer, and that each layer maypossess both control and fluid channels therein and may be in fluidcommunication with one or more different layers through vias.Preferably, the devices described herein may be made of one or moreelastomeric layers, preferably wherein two or more layers are bondedtogether. Layers may be bonded together, preferably by usingcomplimentary chemistries in two or more layers which, when contacted,bond together, or more preferably, wherein one or more layers is treatedwith plasma, preferably Ar plasma, and more preferably, clean dry airplasmas etching prior to bonding, and preferably by bonding with anadhesive, preferably an adhesive comprising components similar orcomplimentary to the chemistry of one or more of the layers being bondedtogether. Adhesives may be applied by spin coating a layer surface, orby spin coating a layer of adhesive onto a surface and stamping a layeron such spun adhesive to apply adhesive to such layer prior to bondingthe layer to another layer.

FIG. 11G depicts a plan view of a preferred embodiment wherein four (4)sets of ninety-six (96) separate metering cells are forming in anelastomeric block.

The extremely small volumes capable of being delivered by pumps andvalves in accordance with the present invention represent a substantialadvantage. Specifically, the smallest known volumes of fluid capable ofbeing manually metered is around 0.1 μl. The smallest known volumescapable of being metered by automated systems is about ten-times larger(1 μl). Utilizing pumps and valves in accordance with the presentinvention, volumes of liquid of 10 nl or smaller can routinely bemetered and dispensed. The accurate metering of extremely small volumesof fluid enabled by the present invention would be extremely valuable ina large number of biological applications, including diagnostic testsand assays.

Equation 1 represents a highly simplified mathematical model ofdeflection of a rectangular, linear, elastic, isotropic plate of uniformthickness by an applied pressure:

w=(BPb ⁴)/(Eh ³), where:

w=deflection of plate;

B=shape coefficient (dependent upon length vs. width and support ofedges of plate);

P=applied pressure;

b=plate width

E=Young's modulus; and

h=plate thickness.

Thus even in this extremely simplified expression, deflection of anelastomeric membrane in response to a pressure will be a function of:the length, width, and thickness of the membrane, the flexibility of themembrane (Young's modulus), and the applied actuation force. Becauseeach of these parameters will vary widely depending upon the actualdimensions and physical composition of a particular elastomeric devicein accordance with the present invention, a wide range of membranethicknesses and elasticity's, channel widths, and actuation forces arecontemplated by the present invention.

It should be understood that the formula just presented is only anapproximation, since in general the membrane does not have uniformthickness, the membrane thickness is not necessarily small compared tothe length and width, and the deflection is not necessarily smallcompared to length, width, or thickness of the membrane. Nevertheless,the equation serves as a useful guide for adjusting variable parametersto achieve a desired response of deflection versus applied force.

The microfluidic devices of the present invention may be used asstand-alone devices, or preferably, may be used as part of a system asprovided for by the present invention. FIG. 12A depicts a perspectiveview of a robotic station for actuating a microfluidic device. Anautomated pneumatic control and accumulator charging station 1200includes a receiving bay 1203 for holding a microfluidic device 1205 ofthe present invention such as the type depicted in FIGS. 8A-G. A platen1207 is adapted to contact an upper face 1209 of microfluidic device1205. Platen 1207 has therein ports that align with microfluidic device1205 to provide fluid pressure, preferably gas pressure, to wells andaccumulators within microfluidic device 1205. In one embodiment, platen1207 is urged against upper face 1221 of microfluidic device 1205 bymovement of an arm 1211, which hinges upon a pivot 1213 and is motivatedby a piston 1215 which is attached at one end to arm 1211 and at theother end to a platform 1217. Sensors along piston 1215 detect pistonmovement and relay information about piston position to a controller,preferably a controller under control of a computer (not shown)following a software script. A plate detector 1219 detects the presenceof microfluidic device 1205 inside of receiving bay 1203, and preferablycan detect proper orientation of microfluidic device 1205. This mayoccur, for example, by optically detecting the presence and orientationof microfluidic device 1205 by reflecting light off of the side ofmicrofluidic device 1205. Platen 1207 may be lowered robotically,pneumatically, electrically, or the like. In some embodiments, platen1207 is manually lowered to engage device 1205.

FIG. 12B depicts charging station 1200 with platen 1207 in the downposition urged against upper face 1221 of microfluidic device 1205,which is now covered by a shroud of platen 1207. In one embodiment,fluid lines leading to platen 1207 are located within arm 1211 and areconnected to fluid pressure supplies, preferably automatic pneumaticpressure supplies under control of a controller. The pressure suppliesprovide controlled fluid pressure to ports within a platen face (notshown) of platen 1207, to supply controlled pressurized fluid tomicrofluidic device 1205. Fine positioning of platen 1207 is achieved,at least in-part, by employing a gimbal joint 1223 where platen 1207attaches to arm 1211 so that platen 1207 may gimbal about an axisperpendicular to upper face 1221 of microfluidic device 1205.

FIGS. 12C and 12D provide side-views of charging station 1200 in both upand down positions, respectively. FIG. 12E depicts a close-up view ofplaten 1207 in a down position.

FIG. 12F depicts a cut-away side-view of platen 1207 urged against upperface 1221 of microfluidic device 1205. Platen 1207 is urged againstupper face 1221 of microfluidic device 1205 to form a fluid tight sealbetween microfluidic device 1205 and a platen face 1227, or betweenportions of device 1205 and face 1227. Platen face 1227, in oneembodiment, includes or is made of a compliant material such as aresilient elastomer, preferably chemical resistant rubber or the like.Inside platen 1207 are separate fluid pressure lines, preferably gaspressure lines, which mate with various locations on upper face 1221 ofmicrofluidic device 1205. Also shown are check valve purge actuators1233 which are actuated, preferably pneumatically, and which whenactuated, push a pin 1231 downward into check valve 812 to open andrelieve fluid pressure, or permit the introduction of fluid throughcheck valve 812 by overcoming its opening resistance. In one embodiment,platen 1207 has first and second purge actuators 1233 which engage checkvalves 811 and 812 (see FIG. 8B).

In another embodiment, chip or device 1205 is manufactured with normallyclosed containment and/or interface valves. In this embodiment,accumlators would not be necessary to hold valves shut duringincubation. Pressure would be applied to carrier or device 1205 wellregions when interface and/or containment valves are desired to beopened. For all or most other times, the valves would remain closed toseparate the various chip experiments from one another, and/or toseparate reagent and protein wells on the chip from one another.

FIG. 12G provides an extreme close-up view of purge actuator 1233 actingupon check valve 812 located within substrate 800 of microfluidic device1205.

FIG. 12H depicts a cut-away view of platen 1207 urged against upper face1221 of microfluidic device 1205 wherein a pressure cavity 1255 isformed above well row 806 by contacting platen face 1227 against a ridge1250 of upper face 1221. Fluid pressure, preferably gas pressure, isthen applied to pressure cavity 1255 by introducing a fluid into cavity1255 from pressure lines running down arm 1211 of charging station 1200.Pressure is regulated by pressure regulators associated with chargingstation 1200, preferably by electronically controlled variable pressureregulators that can change output pressure in accordance with signalssent by a charging station controller, preferably under computercontrol. Fluid pressure inside of pressure cavity 1255 in turn drivesliquid within well 805 through the channels within substrate 800 andinto channels and/or chambers of elastomeric block 808 to fill channelsor chambers or to actuate a deflectable portion of elastomeric block808, preferably a deflectable membrane valve as previously described.

FIG. 13 depicts a rear plan view of the fluidic routing within platenface 1227, and the spatial location of each fluid pressure port ofplaten face 1227 according to a particular embodiment of the presentinvention. In a particular embodiment, fluid interfaces of platen 1207are positioned to be aligned with fluid ports, wells 805, check valvesand the like when platen 1207 engages microfluidic device 1205. In aparticular embodiment, microfluidic device 1205 is an integrated carrierand microfluidic chip such as the Topaz® 1.96 or Topaz® 4.96 chips.

Interrupted diffusion is believed to allow diffusion for a period oftime sufficient to cause the smaller crystallizing agents to diffuseinto the chamber containing protein while limiting the counter diffusionof proteins into the crystallization reagent chamber by closing theinterface valve. The interface valve, when actuated, separates thechamber containing protein from the chamber containing crystallizationreagent.

The present invention provides for devices, systems and methods forusing such devices and systems, for holding and manipulatingmicrofluidic devices, in particular, multilayer elastomeric microfluidicdevices wherein at least one deflectable membrane acts as a valve tointerrupt or separate fluid within a microfluidic channel having across-sectional dimension of about 500 micrometers. Exemplarymicrofluidic devices are used to screen for conditions which causeprotein crystals to form from protein solutions by free-interfacediffusion (FID). In use, the microfluidic devices are loaded with aprotein solution and a crystallization agent, typically in the form of areagent solution, wherein each solution enters into individual chambersinterconnected by a channel having a valve therein. Containment valvesare then used to keep each of the solutions in their respective chamberas the valve located in the channel separating the chambers is opened toinitiate diffusion between the chambers. In preferred devices, thevalves are actuated by changes in fluid pressure, for example eitherhydraulically or pneumatically. Therefore, a means for changing fluidpressure to each of the valve is helpful.

The invention provides, in one aspect, for a carrier that providesaccess to controlled fluid pressure. FIG. 14A depicts a perspective viewof a preferred embodiment. The carrier in FIG. 14A, which in oneembodiment has about a three inch square footprint and is about one inchin height, is preferably made from a polymer, preferably acrylic. Othermaterials may be used depending on the nature of the experiments to beperformed using the carrier, and the solvents that the carrier may beexposed to during use. For example, a carrier could be made frompolypropylene to provide resistance to certain solvents such as acetone.

Turning now to FIGS. 14A and 14B a particular embodiment of the presentinvention will be described. FIG. 14A depicts a carrier 1400 adapted toreceive a microfluidic device or chip (not shown in FIG. 14A), such as achip used to grow protein crystals. The chip is mounted in carrier 1400,integrally formed with carrier 1400, or is a stand alone chip havingsimilar size, features and functions as carrier 1400. In one embodiment,carrier 1400 includes a plurality of ports or wells that are in fluidcommunication with corresponding wells on the microfluidic device. Inthis manner, fluids provided to the carrier wells can in turn bedelivered to the microfluidic device. Further, fluids disposed in thecarrier or device wells can be delivered to testing regions within thedevice by applying pressure to the ports or wells on carrier 1400.

In a particular embodiment, the microfluidic device or chip is receivedin a chip region 1410 disposed in carrier 1400, or integrally formedtherewith. In one embodiment, carrier 1400 includes a first well region1420 and a second well region 1422 adapted to receive a plurality ofreagents. In one embodiment, first well region 1420 and second wellregion 1422 are each adapted to receive up to forty-eight (48) reagentsapiece. In one embodiment, regions 1420 and 1422 comprise a plurality ofwells that are coupled to corresponding wells on the microfluidic devicewhen the device is disposed within carrier 1400. This may occur, forexample, using channels in carrier 1400 as previously described. In oneembodiment, carrier 1400 further includes a first protein region 1430and a second protein region 1432. First protein region 1430 includes aplurality of wells, and in a particular embodiment four wells or ports,adapted to receive desired proteins. In another embodiment, secondprotein region 1432 is adapted to receive up to four proteins. In aparticular embodiment, second protein region 1432 provides vents forcarrier 1400. In other embodiments, the number of wells vary from thosenoted herein for regions 1420, 1422, 1430 and 1432 depending on a widerange of factors including, without limitation, the desired number ofexperiments or tests, the desired well or crystal size, the carriersize, and the like.

In some embodiments it is desirable to control the humidity of the chip.In one embodiment, a hydration chamber 1440 is formed around the chip,with hydration chamber 1440 adapted to hold a fluid or a fluid source.In a particular embodiment, a sponge, a gel package, a woven materialsuch as a piece of cloth or a cotton ball/pad, or other material adaptedto hold a liquid is disposed within hydration chamber 1440. In aparticular embodiment, fluid-containing material may be disposed on bothsides of the chip as indicated in FIG. 14B. The sponge or other materialmay be hydrated with water, buffer, a crystallization reagent, or asolvent. Alternatively, a desiccating material may added to removemoisture from the microfluidic device. Carrier 1400 further includes aninterface accumulator 1460 having a check valve 1465 and a containmentaccumulator 1450 having a check valve 1455. As previously described inconjunction with earlier embodiments, check valves 1455, 1465 areadapted to allow the increase or release of pressure within accumulators1450 and 1460, to introduce or remove fluids from accumulators 1450 and1460, and also to operate to maintain the pressure within carrier 1400,and thus to maintain or apply pressure to appropriate regions of themicrofluidic device disposed therein. The advantage of having an“on-board” source of controlled fluid pressure is that the microfluidicdevice, if actuated by changes in fluid pressure, can be kept in anactuated state independent of an external source of fluid pressure, thusliberating the microfluidic device and carrier from an umbilical cordattached to that external source of fluid pressure. The accumulator mayfurther include a gas pressurization inlet port, a liquid addition port,and a pressurized fluid outlet for communicating fluid pressure to theconnection block.

In a particular embodiment, the integrated carrier 1400 and microfluidicdevice are adapted for performing desired experiments according toembodiments of the present invention by using the systems of the presentinvention. More specifically, as shown in FIG. 15A, a system 1500includes one or more receiving stations 1510 each adapted to receive acarrier 1400. In a particular embodiment, system 1500 includes four (4)receiving stations 1510, although fewer or a greater number of stations1510 are provided in alternative embodiments of the present invention.FIG. 15B depicts carrier 1400 and a device in combination disposed instation 1510 under an interface plate 1520. Interface plate 1520 isadapted to translate downward in FIG. 15B so that interface plate 1520engages the upper surface of carrier 1400 and its microfluidic device.In some embodiments, station 1510 and platen 1520 are similar to station1200 and platen 1207. Interface plate 1520 includes one or more ports1525 for coupling with regions in carrier 1400 which are adapted toreceive fluids, pressure, or the like. In some embodiments, interfaceplate 1520 includes two ports, three ports, four ports, five ports, sixports, seven ports, eight ports, nine ports, ten ports, or the like. Ina preferred embodiment, interface plate 1520 is coupled to six lines forproviding pressure to desired regions of carrier 1400, and two lines forproviding a mechanism for activating check valves 1455 and 1465.

FIG. 15C depicts various regions of interface plate 1520 according to aparticular embodiment of the present invention, similar to FIG. 13. Inalternative embodiments interface plate 1520 includes a different numberor configuration of ports than those depicted in FIG. 15C.

As shown in FIG. 15A, system 1500 further includes a processor that, inone embodiment, is a processor associated with a laptop computer orother computing device 1530. Computing device 1530 includes memoryadapted to maintain software, scripts, and the like for performingdesired processes of the present invention. Further, computing device1530 includes a screen 1540 for depicting results of studies andanalyses of microfluidic devices, with FIG. 17 depicting one embodimentof a screen shot for display on system 1500. System 1500 is coupled toone or more pressure sources, such as a pressurized fluid, gas, or thelike, for delivering same to the microfluidic devices which are fluidlycoupled to interface plate(s) 1520.

FIGS. 16A and 16B depict a particular embodiment of system 1500, andmore specifically, of interface plate 1520. In FIG. 16A, interface plate1520 is coupled to the integrated chip and carrier 1400 in a manner thatfluidly seals certain regions thereof. In particular, fluid seals areprovided between interface plate 1520 and one or more regions of carrier1400 and chip, such as the first protein region 1430, second proteinregion 1432, first well region 1420, second well region 1422, interfaceaccumulator 1460, check valve 1465, containment accumulator 1450, and/orcheck valve 1455. In one embodiment, interface plate 1520 provides fluidseals to regions 1420, 1422, 1430, 1432, and to accumulator 1450 and1460. In one embodiment, interface plate 1520 provides one or more checkvalve actuators 1570 as best seen in FIG. 16B.

In some embodiments, interface plate 1520 provides all of the desiredfluid seals to carrier 1400 and the microfluidic device. In doing so,interface plate 1520 may include a sealing gasket 1580. Sealing gasket1580 may comprise a wide range of materials, including withoutlimitation silicon rubber, an elastomer, or the like. In someembodiments, gasket 1580 comprises a compliant material to help formfluidic seals at the desired locations. In this manner, system 1500 canprovide the desired pressures to appropriate regions of chip and carrier1400. In other embodiments, interface plate 1520 is a two or more platecomponents. For example, the regions or ports on carrier 1400 and themicrofluidic device each may be fluidly coupled to a separate plate 1520adapted to fit that port or region. System 1500 then would include thenecessary number of interface plates 1520 for the various ports orregions. Further, in some embodiments, more than one region or port iscoupled to a particular interface plate 1520, while other regions orports are coupled to a separate interface plate 1520. Other combinationsof interface plates and carrier/chip regions and ports also fall withinthe scope of the present invention.

The operation of system 1500, in one embodiment, involves the loading ofone or more carriers 1400 into receiving station(s) 1510. In someembodiments, carriers 1400 include the microfluidic device coupledthereto, and have desired reagents and proteins loaded into the carrierwells prior to placing the carriers into receiving stations 1510. Inother embodiments, the carriers 1400 are placed into receiving stations1510, and subsequently loaded with reagents and proteins. Carriers 1400further may be loaded with a hydration fluid. Hydration fluid may beplaced in hydration chamber 1440. After carriers 1400 are loaded intosystem 1500, interface plates 1520 are lowered or otherwise translatedto engage carriers 1400. Plates 1520 may be manually, robotically, orotherwise lowered to fluidly seal with portions or all of chip/carrier1400. A hydration fluid is provided to interface accumulator 1460 and/orcontainment accumulator 1450 and is driven into the chip by applying theappropriate pressure to accumulators 1450, 1460 using a pressure sourcecoupled to interface plate 1520. In a particular embodiment, system 1500automatically performs this process, which in a particular embodimentoccurs within about twenty (20) hours after the hydration fluid is addedto carrier 1400. As a result, the chip is sufficiently loaded withhydraulic fluid to operate chip containment and/or interface valves, asdescribed herein and more fully in the patents and applicationspreviously incorporated herein by reference.

The proteins and reagents are dispensed into the chip by applying thedesired pressure to the appropriate sealed chip regions around theappropriate inlets. For example, applying a pressure between about 1 psiand about 35 psi to first and second well regions 1420 and 1422 operatesto drive the reagents into the chip. Similarly, applying a pressurebetween about 1 psi and about 35 psi to first and second protein regions1430, 1432 operates to drive the proteins into the chip. In a particularembodiment, this occurs within about sixty (60) minutes after loadingthe chip with hydraulic fluid. Once the proteins and reagents have beendriven to the desired wells, chambers, reservoirs or the like within thechip, the interface valves within the chip are opened by releasing checkvalve 1465 in interface accumulator 1460. In a particular embodiment,check valve 1465 is released, to open interface valves in the chip, whensystem 1500 activates check valve actuator 1570 which engages checkvalve 1465. In some embodiments, check valve actuator 1570 includes apin, a post, or the like adapted to engage check valve 1465 in order torelease pressure within interface accumulator 1460. In an alternativeembodiment, check valve 1465 is manually released or opened.

After the reagent and proteins are allowed to mix for a desired periodof time, using free interface diffusion or other appropriate processes,the interface valves are closed. A pressure is applied to actuators 1450and/or 1460 in order to maintain closed interface valves and containmentvalves. The carrier 1400 may be removed from system 1500 for incubationor storage. Actuators 1450 and 1460 hold the pressure for a desiredperiod of time, from hours to days, in order to prevent or help preventthe containment and interface valves from opening. In a particularembodiment, actuators 1450 and 1460 maintain the pressure within thechip above a desired threshold pressure sufficient to keep containmentand/or interface valves closed. In one embodiment, actuators 1450 and1460 maintain the pressure above the threshold pressure for at least two(2) days, at least seven (7) days, and the like. The length of timeactuators 1450 and 1460 maintain the desired pressure depends in part onthe incubation temperature. Depending in part on the desired incubationperiod length and/or incubation conditions, carrier 1400 may be returnedto system 1500 in order to recharge or repressurize actuators 1450,1460. In this manner, the incubation period may be extended to helpprovide for desired crystal growth or other chemical or relatedprocesses.

FIG. 17 depicts a typical graphical user interface computer screengenerated by a computer driving station 1510 as described above. In apreferred embodiment which is shown, four different charging stationscan be actuated independently, as shown by the four separate screencolumns indicating status. The software can be linked to a data inputdevice and a database to correlate experimental conditions, reagentsused, user identification, sample character, valve actuation profiles,humidity, and post reaction analysis data by associating a uniqueidentifier, preferably a bar or spatial dot optical code or an encodedradio frequency device with a microfluidic device of the presentinvention. Information may be generated by different laboratoryinstruments, such as robotic dispensing stations, robotic platehandlers, incubators, charging stations as described herein, and opticalinspection stations, such as those described in copending U.S.Provisional Patent Application No. 60/472,226 by Lee et al filed on May20, 2003, 60/490,712 and 60/490,584, both by Taylor, and 60/490,666 byQuan, each of the three filed on Jul. 28, 2003 and are all commonlyassigned to the assignee of the present application, and which are eachherein incorporated by reference in their entireties for all purposes.

Software used to operate the charging stations described herein mayfurther provide for an end-user script writing feature which allows anend user to write custom scripts to actuate and otherwise control ormanipulate the microfluidic devices described herein. Such customscripts may further integrate with other computer programs and computercontrolled devices used in experiments involving the microfluidicdevices of the present invention.

Example 1: In a preferred embodiment, a protein crystallizationreactions may be carried out by controlling diffusion by closing theinterface valve after a period of time, for example, after 60 minutes.Table 1, below, highlights the steps for using an exemplary proteincrystallization device of the invention in a manner for which diffusionis interrupted after a period of time.

TABLE 1 Script Name Time Description 1_Prime 1 min. Fills interface andcontainment lines with control line fluid and closes control linevalves. Allows a pause to inspect valve closure. The last step opensinterface valves. Use to prepare 1.96 Chip for experiment setup and testaccumulator pressurization. 2_Load 1.96 20 min. Closes containmentvalves, pressurizes reagent and protein blindfill up to containmentvalves, closes interface valves, opens containment valves, continuesloading protein and reagent up to interface valve, closes containmentvalve. Chip is ready for T0 scan at end of script. 2-5_Load 120 min.Merges 2_Load 1.96 with 5_Ctrld FID 60 min. Use in place of the twoscripts. Use to blind fill reagents, begin diffusion, and then stop FIDafter to min. Use if T0 scan is not needed. 2-52 4C Load 100 min. Merges2_Load 1.96 5_2 4C Ctrld FID 100 min. Use in place of the two scriptsUse at 4° C. after 1_Prime to load reagents and protein. Chip is readyfor T0 scan at end of script. 3_Reload 1.96 16 min. Closes interfacevalves, pressurizes reagent and protein blindfill up to interfacevalves, closes containment valves. Use if wells are not completelyfilled at the end of the 2_Load 1.96 script. Chip is ready for T0 scanat end of script. 2_53 13C Load 100 min. Merges 2_Load 1.96 5_2 13CCtrld FID 80 min. Use in place of the two scripts. Use at 13° C. after1_Prime to load reagents and protein. Chip is ready for T0 scan at endof script. 4_Post T0 30 sec. Opens interface valves to begin diffusion.Use after T0 scan. Chip is ready for incubation at end of script.5_Controlled 60.5 min. Opens interface valves to begin diffusion, then,after a 60-min. FID 60 min period of diffusion, closes interface valvesand recharges containment accumulator. Use after T0 scan as analternative to 4_Post T0 to begin diffusion and then interrupt FID after60 min. 5_2 4C Ctrld 100 min. Opens interface valves to begin diffusion.After a 100-min. period of FID 100 min diffusion, closes interfacevalves and recharges containment accumulator. Use at 4° C. after T0 scanas an alternative to 4_Post T0 to begin diffusion and then interrupt FIDafter 100 min. 5_2 13C Ctrld 80 min. Opens interface valves to begindiffusion. After a 80-min. period of FID 80 min diffusion, closesinterface valves and recharges containment accumulator. Use at 4° C.after T0 scan as an alternative to 4_Post T0 to begin diffusion and theninterrupt FID after 80 min. 5_2_4C Ctrld 2.5 hr. Opens interface valvesto begin diffusion, then, after a 90-min. FID period of diffusion,closes interface valves and recharges containment accumulator. Use at 4°C., after T0 scan as to begin diffusion and then interrupt FID after 60min. 5_3_13C Ctrld 2 hr. Opens interface valves to begin diffusion,then, after a 90-min. FID period of diffusion, closes interface valvesand recharges containment accumulator. Use at 4° C., after T0 scan as tobegin diffusion and then interrupt FID after 90 min. 6_Recharge 30 sec.Recharges interface and containment accumulator. Use every 24 hr. duringincubation.

In some embodiments, the integrated chip carrier (ICC) and elastomericchip attached thereto, as described generally herein, are used tofacilitate polymerase chain reactions (PCR). However, when attemptingPCR using a PCR chip, the thermal conductivity of a plastic ICC may noteffectively create a homogeneous thermal field among the array ofreactions harbored within the elastomeric chip. For example, the ICCdepicted in FIG. 18A, while useful for protein crystallization amongother processes, may not transfer heat sufficiently uniformly to thechip coupled thereto. Further, the operation of the ICC of FIG. 18A mayrequire it be retained against a platen. The application of acompressive force, as shown in FIG. 19A, can negatively affect the chip.Therefore, in some embodiments of the present invention, improvedhomogenous thermal fields are provided using the ICC embodimentsdescribed in conjunction with FIG. 18B, and improved ICC retentionmethods are depicted in FIG. 19B.

In some embodiments, an elastomeric chip is designed such that fluidicconnections with the ICC are located at the outer boundaries of theelastomer chip. In this manner, a portion of the ICC need not be reliedupon for fluidic transport. One such embodiment of an ICC 1800 isdepicted in FIG. 18B. In some embodiments, the region of the elastomericchip that is not in contact with the ICC surface is where the array ofreaction chambers is located. This reaction area 1810 includes theelastomeric chip and a thermal conductive material that is mated againstthe underside of the chip (the side of the elastomeric block that wouldhave otherwise contacted the plastic portion of the ICC). The size andshape of reaction area 1810 may vary within the scope of the presentinvention, with one embodiment depicted in FIG. 19C showing therelationship between the ICC and chip.

In this manner, thermal energy (e.g., from a PCR machine) can betransmitted to the elastomeric block with minimal or reduced thermalimpedance. In some embodiments, the thermal conductive materialcomprises silicon (Si). In a particular embodiment, silicon frompolished and smooth silicon wafers, similar to or the same as that usedin the semiconductor industry, are used. Other low thermal impedancematerials also may be used within the scope of the present invention,depending on the nature of the thermal profiles sought. In someembodiments, the thermal conductive material has low thermal mass (i.e.,materials that effect rapid changes in temperature, even though a goodthermal conductor, e.g. copper). In some embodiments, polished siliconis used to enhance mirroring effects and increase the amount of lightthat can be collected by the detector used in the system, either in realtime, or as an end-point analysis of the PCR reaction. These benefitsmay also improve iso-thermal reactions.

With ICC 1800, one wishing to perform PCR may do so by mating thereaction region of the elastomeric block with a source of thermalcontrol. The thermal control source may include a PCR machine, a heatedplaten, a separate heat source, among others. In some embodiments, theICC fits into a standard PCR machine that accepts flat bottom reactionplates, and/or has a flat thermal plate wherein adapters for variousformats of tube-based PCR may be used. However, each of thosearrangements rely generally on compression of the plate downward toachieve good (homogeneous) thermal contact, as shown in FIG. 19A. Insome embodiments of the present invention, the elastomeric chips are notamenable to downward compression because the flexible valves andelastomeric chamber may deform and cause undesired fluidic actionswithin the elastomeric chip.

In other embodiments, the negative effects of downward compression ofthe ICC are reduced or avoided by using a vacuum chuck to mate againstthe thermal material bonded to the underside of the otherwise exposedelastomeric block. In this manner, when a vacuum is applied to thechuck, a tight seal is created between the vacuum chuck and the thermalmaterial of the ICC. In one embodiment as shown in FIG. 19B, a vacuumchuck 1950 comprises or is made from one or more materials that are goodthermal conductors which are bonded to a source of thermal control,e.g., one or more Peltier devices. In one embodiment, a plurality ofthermal controls are used to generate thermal gradients among the arrayof reactions.

In use, the ICC is positioned over vacuum chuck 1950 and lowered down,or otherwise translated, to contact the thermal portion 1920 of theintegrated chip 1910 and ICC 1930 with vacuum chuck 1950. Again, in oneembodiment, thermal portion 1920 comprises silicon. Thermal portion 1920is depicted coupled to elastomeric block or chip 1910, with suchcoupling being effected using adhesive or the like in some embodiments.As shown, block 1910 engages ICC 1930, and in one embodiment a gap 1940is maintained between thermal portion 1920 and ICC 1930. Gap 1940 helpsthermally isolate ICC 1930 from a thermal heat source, such as chuck orplaten 1950. Additionally, in one embodiment gap 1940 permits someflexure of block 1910 and/or thermal portion 1920. In this manner, gap1940 in some embodiments helps form a seal when a vacuum is applied tochuck 1950 through one or more vacuum ports 1960 to pull the thermalportion 1920 towards chuck 1950. The amount of vacuum achieved may bemonitored to verify that good thermal contact has indeed been madebetween chuck 1950 and thermal portion 1920. In some embodiments, one ormore ports in the vacuum chuck are used for monitoring the level ofvacuum. In a particular embodiment as shown in FIG. 19D and FIG. 20, oneor more ports used for monitoring the level of vacuum would be locateddistal to the vacuum ports and in fluidic communication with each otherthrough a path, preferable a tortuous and narrow path. In this manner,the differential between one side of the vacuum chuck-thermal portion ofthe ICC can be compared to the other side of the vacuum chuck-thermalportion of the ICC to determine whether an attempt to re-place the ICCon the vacuum chuck may resolve vacuum loss problems, if any. This couldbe an iterative process either done automatically or manually, or somecombination thereof.

FIG. 20 depicts one embodiment of a chuck according to the presentinvention. The nature of the tortuous path used to achieve an accuratemeasurement of differential vacuum levels across the vacuum chuckthermal portion area, and to foster a homogeneous, or relativelyhomogeneous application of vacuum to the thermal portion of the ICC, mayvary within the scope of the present invention. By using this approach,the elastomeric block used to carry out the PCR reactions can be in goodthermal contact with a PCR machine without jeopardizing thefunctionality of the elastomeric (deflectable) portions of theelastomeric block if ordinary downward compression would be used.

While the present invention has been described herein with reference toparticular embodiments thereof, a latitude of modification, variouschanges and substitutions are intended in the foregoing disclosure, andit will be appreciated that in some instances some features of theinvention will be employed without a corresponding use of other featureswithout departing from the scope of the invention as set forth. Forexample, in addition to pressure based actuation systems describedabove, optional electrostatic and magnetic actuation systems are alsocontemplated. It is also possible to actuate the device by causing afluid flow in the control channel based upon the application of thermalenergy, either by thermal expansion or by production of gas from liquid.Further, in another embodiment, centrifugal force are used to driveprotein and reagents into the chip. Therefore, many modifications may bemade to adapt a particular situation or material to the teachings of theinvention without departing from the essential scope and spirit of thepresent invention. It is intended that the invention not be limited tothe particular embodiment disclosed as the best mode contemplated forcarrying out this invention, but that the invention will include allembodiments and equivalents falling within the scope of the claims.

The disclosure set forth above may encompass one or more distinctinventions, with independent utility. Each of these inventions has beendisclosed in its preferred form(s). These preferred forms, including thespecific embodiments thereof as disclosed and illustrated herein, arenot intended to be considered in a limiting sense, because numerousvariations are possible. The subject matter of the inventions includesall novel and nonobvious combinations and subcombinations of the variouselements, features, functions, and/or properties disclosed herein.

1. A method of conducting a reaction at a selected temperature or rangeof temperatures over time, the method comprising: providing an arraydevice for containing a plurality of separate reaction chambers, thearray device comprising an elastomeric block formed from a plurality oflayers, wherein at least one layer has at least one recess formedtherein, the recess having at least one deflectable membrane integral tothe layer with the recess, the array device further comprising a thermaltransfer device proximal to at least one of the reaction chambers, thethermal transfer device being formed to contact a thermal controlsource; introducing into the array device reagents for carrying out adesired reaction; and contacting the array device with a thermal controldevice such that the thermal control device is in thermal communicationwith the thermal control source so that a temperature of the reaction inat least one of the reaction chambers is changed as a result of a changein temperature of the thermal control source. 2-5. (canceled)
 6. Themethod recited in claim 1 wherein the thermal control device is adaptedto apply a force to the thermal transfer device to urge the thermaltransfer device towards the thermal control source.
 7. The methodrecited in claim 6 wherein the force comprises a magnetic,electrostatic, or vacuum force.
 8. The method recited in claim 7 whereinthe force comprises a vacuum force applied towards the thermal transferdevice through channels formed in a surface of the thermal controldevice or the thermal transfer device.
 9. The method recited in claim 8further comprising detecting a level of vacuum achieved between thesurface of the thermal control device and a surface of the thermaltransfer device. 10-15. (canceled)
 16. A device comprising the arraydevice described in claim
 1. 17. A unit comprising the thermal controldevice as described in claim
 9. 18. A system comprising the array deviceof claim 1 and the thermal control device of claim
 9. 19. A microfluidicsystem comprising: an array device for containing a plurality ofseparate reaction chambers disposed within a reaction area and in fluidcommunication with fluid inlets to the array device disposed outside thereaction area, the array device comprising an elastomeric block formedfrom a plurality of layers, wherein at least one layer has at least onerecess formed therein, the recess having at least one deflectablemembrane integral to the layer with the recess; a carrier adapted tohold the array device and having a plurality of fluid channelsinterfaced with the fluid inlets; and a thermal transfer interfacecomprising a thermally conductive material disposed to providesubstantially homogeneous thermal communication from a thermal controlsource to the reaction area.
 20. The microfluidic system recited inclaim 19 wherein the thermally conductive material is reflective. 21.The microfluidic system recited in claim 19 wherein the thermallyconductive material comprises a semiconductor.
 22. The microfluidicsystem recited in claim 19 wherein the thermally conductive materialcomprises silicon.
 23. The microfluidic system recited in claim 19wherein the thermally conductive material comprises polished silicon.24. The microfluidic system recited in claim 19 wherein the thermallyconductive material comprises a metal.
 25. The microfluidic systemrecited in claim 19 wherein the reaction area is located within acentral portion of the array device and the fluid inlets are disposed ata periphery of the array device.
 26. The microfluidic system recited inclaim 25 wherein the array device is coupled with the carrier at theperiphery of the array device and the thermally conductive material iscoupled with a surface of the array device at the reaction area.
 27. Themicrofluidic system recited in claim 19 further comprising means forapplying a force to the thermal transfer interface to urge the thermaltransfer interface towards the thermal control source.
 28. Themicrofluidic system recited in claim 27 wherein the means for applying aforce comprises a means for applying a vacuum source towards the thermaltransfer interface through channels formed in a surface of a thermalcontrol device or in the thermal transfer device.
 29. The microfluidicsystem recited in claim 28 further comprising a vacuum level detectorfor detecting a level of vacuum achieved between the surface of thethermal control device and a surface of the thermal transfer device. 30.The microfluidic system recited in claim 29 wherein the vacuum leveldetector is located at a position along the channel or channels distalfrom a location of a source of vacuum.
 31. The microfluidic systemrecited in claim 19 further comprising an automatic control system inoperable communication with a robotic control system for introducing andremoving the array device from the thermal control source.